Leptospirosis is among the world's most important zoonotic infectious diseases, annually affecting an estimated 868,000 people globally, with an estimated case fatality rate of 5-10%. Important gaps remain in translating recent fundamental molecular advances in studies of Leptospira and leptospirosis4 to public health approaches to preventing and ameliorating this infectious disease. The long-term goal of this project is to develop and validate new tools and approaches to reduce the impact of leptospirosis on human health. The studies proposed here will test the central hypothesis that human risk for acquiring leptospirosis and clinical outcomes of infection can be predicted by quantifying and identifying infectious Leptospira (pathogenic and intermediately pathogenic species and serovars) in environmental surface waters and soils (most important sources of human infection) using metagenomic and deep sequencing tools. This hypothesis will be tested in three Specific Aims: 1) To determine the incidence of acute leptospirosis, and the prevalence, duration and clinical consequences of Leptospira renal carriage in prospective, observational, population- based cohort studies in urban and rural areas of Iquitos, Peru; 2) To quantify, identify and determine the local distribution of infectious Leptospira in likely environmental sources of transmission in leptospirosis-endemic urban and rural communities of Iquitos, Peru; and 3) To determine whether the urban vs. rural environmental contexts in which Leptospira are found, quantified and typed can predict risk for humans for acquiring severe vs. non-severe leptospirosis. Using recently developed techniques, the natural history and clinical outcomes of highly endemic leptospiral infection will be studied, for the first time, in observational, population-based, prospective cohort studies in contrasting epidemiological settings in the Peruvian Amazon. The patho-biological significance of chronic renal infection will be determined using urinary biomarkers that indicate renal tubular structural damage. The quantity and Leptospira species/serovars in potential environmental sources of transmission will be determined using metagenomic techniques. This multidisciplinary project will provide key data to drive the development of public health policies aimed at reducing the human risk of leptospirosis. The study results will be globally applicable to other regions affected by leptospirosis, and have the potential to be generalizable to other infectious diseases that have key eco/epidemiological contexts, such as those caused by enteric pathogens. For the first time, the natural history and clinical impact of leptospirosis in a highly endemic setting will be precisely determined at a population level, essential data for accurately estimating the burden of this globally-important zoonotic disease. Ultimately, this project will validate whether environmental surveillance can be deployed as a public health tool for identifying and eliminating sources of leptospirosis transmission.